Maturation and Conformational Switching of a De Novo Designed Phase-Separating Polypeptide.

Cellular compartments formed by biomolecular condensation are widespread features of cell biology. These organelle-like assemblies compartmentalize macromolecules dynamically within the crowded intracellular environment. However, the intermolecular interactions that produce condensed droplets may also create arrested states and potentially pathological assemblies such as fibers, aggregates, and gels through droplet maturation. Protein liquid-liquid phase separation is a metastable process, so maturation may be an intrinsic property of phase-separating proteins, where nucleation of different phases or states arises in supersaturated condensates. Here, we describe the formation of both phase-separated droplets and proteinaceous fibers driven by a de novo designed polypeptide. We characterize the formation of supramolecular fibers in vitro and in bacterial cells. We show that client proteins can be targeted to the fibers in cells using a droplet-forming construct. Finally, we explore the interplay between phase separation and fiber formation of the de novo polypeptide, showing that the droplets mature with a post-translational switch to largely ß conformations, analogous to models of pathological phase separation.

The Healthspan Project: A Retrospective Pilot of Biomarkers and Biometric Outcomes after a 6-Month Multi-Modal Wellness Intervention.

Wellness-centric proactive healthcare is increasingly sought after, with individuals frequently embracing complementary modalities to achieve this goal. In this six-month study, healthy adult participants (n = 25) received specific therapies, including whole-body cryotherapy, infrared sauna, and photobiomodulation, along with guidance on physical activity, diet, and alcohol intake. Serum biomarkers were measured for all participants, while a subset also received biometric assessments for body composition (n = 10) and heart rate variability (n = 7). Over the course of the study (mean (SD) follow-up days = 174 (130)), participants exhibited significant improvements in health. LDL cholesterol (-9.77 (15.43) md/dL) and hsCRP (-1.75 (2.66) mg/L) decreased significantly (p < 0.05). HbA1c increased slightly (p < 0.05), but the effect size was small (0.12 (0.13)%). The body composition subset lost overall body weight (-3.29 (3.75) kg), primarily body fat, while preserving lean muscle mass (p < 0.05). Heart rate variability increased for those with existing cardiovascular risk factors (p < 0.05). In conclusion, participation in the multimodal Healthspan protocol is associated with substantial improvements in health-related biomarkers and biometrics.

Effect of nanoparticle loading and magnetic field application on the thermodynamic, optical, and rheological behavior of thermoresponsive polymer solutions.

Although processing via external stimuli is a promising technique to tune the structure and properties of polymeric materials, the impact of magnetic fields on phase transitions in thermoresponsive polymer solutions is not well-understood. As nanoparticle (NP) addition is also known to impact these thermodynamic and optical properties, synergistic effects from combining magnetic fields with NP incorporation provide a novel route for tuning material properties. Here, the thermodynamic, optical, and rheological properties of aqueous poly(N-isopropyl acrylamide) (PNIPAM) solutions are examined in the presence of hydrophilic silica NPs and magnetic fields, individually and jointly, via Fourier-transform infrared spectroscopy (FTIR), magneto-turbidimetry, differential scanning calorimetry (DSC), and magneto-rheology. While NPs and magnetic fields both reduce the phase separation energy barrier and lower optical transition temperatures by altering hydrogen bonding (H-bonding), infrared spectra demonstrate that the mechanism by which these changes occur is distinct. Magnetic fields primarily alter solvent polarization while NPs provide PNIPAM-NP H-bonding sites. Combining NP addition with field application uniquely alters the solution environment and results in field-dependent rheological behavior that is unseen in polymer-only solutions. These investigations provide fundamental understanding on the interplay of magnetic fields and NP addition on PNIPAM thermoresponsivity which can be harnessed for increasingly complex stimuli-responsive materials.

Identification and characterisation of a rare MTTP variant underlying hereditary non-alcoholic fatty liver disease.

Background & Aims: Non-alcoholic fatty liver disease (NAFLD) is a complex trait with an estimated prevalence of 25% globally. We aimed to identify the genetic variant underlying a four-generation family with progressive NAFLD leading to cirrhosis, decompensation, and development of hepatocellular carcinoma in the absence of common risk factors such as obesity and type 2 diabetes. Methods: Exome sequencing and genome comparisons were used to identify the likely causal variant. We extensively characterised the clinical phenotype and post-prandial metabolic responses of family members with the identified novel variant in comparison with healthy non-carriers and wild-type patients with NAFLD. Variant-expressing hepatocyte-like cells (HLCs) were derived from human-induced pluripotent stem cells generated from homozygous donor skin fibroblasts and restored to wild-type using CRISPR-Cas9. The phenotype was assessed using imaging, targeted RNA analysis, and molecular expression arrays. Results: We identified a rare causal variant c.1691T>C p.I564T (rs745447480) in MTTP, encoding microsomal triglyceride transfer protein (MTP), associated with progressive NAFLD, unrelated to metabolic syndrome and without characteristic features of abetalipoproteinaemia. HLCs derived from a homozygote donor had significantly lower MTP activity and lower lipoprotein ApoB secretion than wild-type cells, while having similar levels of MTP mRNA and protein. Cytoplasmic triglyceride accumulation in HLCs triggered endoplasmic reticulum stress, secretion of pro-inflammatory mediators, and production of reactive oxygen species. Conclusions: We have identified and characterised a rare causal variant in MTTP, and homozygosity for MTTP p.I564T is associated with progressive NAFLD without any other manifestations of abetalipoproteinaemia. Our findings provide insights into mechanisms driving progressive NAFLD. Impact and Implications: A rare genetic variant in the gene MTTP has been identified as responsible for the development of severe non-alcoholic fatty liver disease in a four-generation family with no typical disease risk factors. A cell line culture created harbouring this variant gene was characterised to understand how this genetic variation leads to a defect in liver cells, which results in accumulation of fat and processes that promote disease. This is now a useful model for studying the disease pathways and to discover new ways to treat common types of fatty liver disease.

Mortality in ASIA Impairment Scale grade A to D Patients With Odontoid Fracture and Magnetic Resonance Imaging Evidence of Spinal Cord Injury.

Odontoid fractures are common, often presenting in the elderly after a fall and infrequently associated with traumatic spinal cord injury (tSCI). The goal of this study was to analyze predictors of mortality and neurological outcome when odontoid fractures were associated with signal change on magnetic resonance imaging (MRI) at admission. Over an 18-year period (2001-2019), 33 patients with odontoid fractures and documented tSCI on MRI were identified. Mean age was 65.3 years (standard deviation [SD] = 17.2), and 21 patients were male. The mechanism of injury was falls in 25 patients, motor vehicle accidents in 5, and other causes in 3. Mean Injury Severity Score (ISS) was 40.5 (SD = 30.2), Glasgow Coma Scale (GCS) score was 13 (SD = 3.4), and American Spinal Injury Association (ASIA) motor score (AMS) was 51.6 (SD = 42.7). ASIA Impairment Scale (AIS) grade was A, B, C, and D in 9, 2, 3, and 19 patients, respectively. Mean intramedullary lesion length was 32.3 mm (SD = 18.6). The odontoid peg was displaced ventral or dorsal in 15 patients. Twenty patients had surgical intervention: anterior odontoid screw fixation in 7 and posterior spinal fusion in 13. Eleven (33.3%) patients died in this series: withdrawal of medical care in 5; anoxic brain injury in 4; and failure of critical care management in 2. Univariate logistic regression indicated that GCS score (p < 0.014), AMS (p < 0.002), AIS grade (p < 0.002), and ISS (p < 0.009) were risk factors for mortality. Multi-variate regression analysis indicated that only AMS (p < 0.002) had a significant relationship with mortality when odontoid fracture was associated with tSCI (odds ratio, 0.963; 95% confidence interval, 0.941-0.986).

Correction: Back et al. A New Micromonospora Strain with Antibiotic Activity Isolated from the Microbiome of a Mid-Atlantic Deep-Sea Sponge. Mar. Drugs 2021, 19, 105.

After publication of this article [...].

Rates and Predictors of Surgery for Lumbar Disc Herniation Between the Military and Civilian Health Care Systems.

STUDY DESIGN: Retrospective review (level of evidence III). OBJECTIVE: Surgical care patterns for lumbar disc herniation (LDH), a common musculoskeletal condition of high relevance to the Military Health System (MHS), have not been described or compared across the direct care and purchased care MHS components. This study aimed to describe surgery rates in MHS beneficiaries who were diagnosed with LDH in direct care versus purchased care and to evaluate characteristics associated with the location of surgery. Differences in care patterns for LDH may suggest unexpected variation within the centrally managed MHS. METHODS: We described 1-year rates of surgery among beneficiaries who were diagnosed with LDH in direct care versus purchased care. Among beneficiaries who were diagnosed in direct care and had surgery, multivariable logistic regression models were used to identify characteristics associated with surgery location. RESULTS: We identified 726,638 MHS beneficiaries who were diagnosed with LDH in direct care or purchased care during the 9-year study period. One-year surgery rates were 10.1% in beneficiaries who were diagnosed in direct care versus 11.3% in beneficiaries who were diagnosed in purchased care. Among the 7467 patients who were diagnosed in direct care and had surgery within 1 year, characteristics associated with lower probability of surgery in purchased care versus direct care included diagnosing facility type (hospital with a neurosurgery or spine specialty versus clinic (odds ratio [OR], 0.12 (95% CI, 0.10-0.15)), Navy versus Army (OR, 0.24 (95% CI, 0.21-0.28)), and diagnosing facility specialty (Medical Expense and Performance Reporting System) (surgical care (OR, 0.33 (95% CI, 0.27-0.40)) and orthopedic care (OR, 0.39 (95% CI, 0.33-0.46)) versus primary care. The presence of comorbidities was associated with higher probability of surgery in purchased care versus direct care (OR, 1.20 (95% CI, 1.06-1.36)). CONCLUSIONS: The 1-year rate of surgery for LDH was modestly higher in beneficiaries who were diagnosed in purchased care versus direct care. Among patients who were diagnosed in direct care, several patient-level and facility-level characteristics were associated with receiving surgery in purchased care, suggesting potentially unexpected variation in care utilization across components of the MHS.

Importance of Prospective Registries and Clinical Research Networks in the Evolution of Spinal Cord Injury Care.

Only 100 years ago, traumatic spinal cord injury (SCI) was commonly lethal. Today, most people who sustain SCI survive with continual efforts to improve their quality of life and neurological outcomes. SCI epidemiology is changing as preventative interventions reduce injuries in younger individuals, and there is an increased incidence of incomplete injuries in aging populations. Early treatment has become more intensive with decompressive surgery and proactive interventions to improve spinal cord perfusion. Accurate data, including specialized outcome measures, are crucial to understanding the impact of epidemiological and treatment trends. Dedicated SCI clinical research and data networks and registries have been established in the United States, Canada, Europe, and several other countries. We review four registry networks: the North American Clinical Trials Network (NACTN) SCI Registry, the National Spinal Cord Injury Model Systems (SCIMS) Database, the Rick Hansen SCI Registry (RHSCIR), and the European Multi-Center Study about Spinal Cord Injury (EMSCI). We compare the registries' focuses, data platforms, advanced analytics use, and impacts. We also describe how registries' data can be combined with electronic health records (EHRs) or shared using federated analysis to protect registrants' identities. These registries have identified changes in epidemiology, recovery patterns, complication incidence, and the impact of practice changes such as early decompression. They've also revealed latent disease-modifying factors, helped develop clinical trial stratification models, and served as matched control groups in clinical trials. Advancing SCI clinical science for personalized medicine requires advanced analytical techniques, including machine learning, counterfactual analysis, and the creation of digital twins. Registries and other data sources help drive innovation in SCI clinical science.

Tuning the thermodynamic, optical, and rheological properties of thermoresponsive polymer solutions via silica nanoparticle shape and concentration.

HYPOTHESIS: The shape and quantity of hydrophilic silica nanoparticles (NPs) can be used to tune the microstructure, rheology, and stability of phase-separating polymer solutions. In thermoresponsive polymer systems, silica nanospheres are well-studied whereas anisotropic NPs have little literature precedent. Here, we hypothesize that NP shape and concentration lower the onset of rheological and turbidimetric transitions of aqueous poly(N-isopropyl acrylamide) (PNIPAM) solutions. EXPERIMENTS: Differential scanning calorimetry (DSC), Fourier-transform infrared spectroscopy (FTIR), turbidimetry, and oscillatory rheology are utilized to examine interactions between NPs, PNIPAM, and water and to track changes in phase separation and rheological properties due to NP concentration and shape. FINDINGS: NP addition reduces phase separation enthalpy due to PNIPAM-NP hydrogen bonding interactions, the degree to which depends on polymer content. While NP addition minorly impacts thermodynamic and optical properties, rheological transitions and associated rheological properties are dramatically altered with increasing temperature, and depend on NP quantity, shape, and polymer molecular weight. Thus NP content and shape can be used to finely tune transition temperatures and mechanical properties for applications in stimuli-responsive materials.

Rationale and Methods for Updated Guidelines for the Management of Penetrating Traumatic Brain Injury.

Penetrating traumatic brain injury (pTBI) affects civilian and military populations resulting in significant morbidity, mortality, and healthcare costs. No up-to-date and evidence-based guidelines exist to assist modern medical and surgical management of these complex injuries. A preliminary literature search revealed a need for updated guidelines, supported by the Brain Trauma Foundation. Methodologists experienced in TBI guidelines were recruited to support project development alongside two cochairs and a diverse steering committee. An expert multi-disciplinary workgroup was established and vetted to inform key clinical questions, to perform an evidence review and the development of recommendations relevant to pTBI. The methodological approach for the project was finalized. The development of up-to-date evidence- and consensus-based clinical care guidelines and algorithms for pTBI will provide critical guidance to care providers in the pre-hospital and emergent, medical, and surgical settings.

Comprehensive phenotyping of cutaneous afferents reveals early-onset alterations in nociceptor response properties, release of CGRP, and hindpaw edema following spinal cord injury.

Spinal cord injury (SCI) is a complex syndrome that has profound effects on patient well-being, including the development of medically-resistant chronic pain. The mechanisms underlying SCI pain have been the subject of thorough investigation but remain poorly understood. While the majority of the research has focused on changes occurring within and surrounding the site of injury in the spinal cord, there is now a consensus that alterations within the peripheral nervous system, namely sensitization of nociceptors, contribute to the development and maintenance of chronic SCI pain. Using an ex vivo skin/nerve/DRG/spinal cord preparation to characterize afferent response properties following SCI, we found that SCI increased mechanical and thermal responding, as well as the incidence of spontaneous activity (SA) and afterdischarge (AD), in below-level C-fiber nociceptors 24 hr following injury relative to naïve controls. Interestingly, the distribution of nociceptors that exhibit SA and AD are not identical, and the development of SA was observed more frequently in nociceptors with low heat thresholds, while AD was found more frequently in nociceptors with high heat thresholds. We also found that SCI resulted in hindpaw edema and elevated cutaneous calcitonin gene-related peptide (CGRP) concentration that were not observed in naïve mice. These results suggest that SCI causes a rapidly developing nociceptor sensitization and peripheral inflammation that may contribute to the early emergence and persistence of chronic SCI pain.

The C5a-C5aR1 complement axis is essential for neutrophil recruitment to draining lymph nodes via high endothelial venules in cutaneous leishmaniasis.

Neutrophils are specialized innate immune cells known for their ability to fight pathogens. However, the mechanisms of neutrophil trafficking to lymph nodes are not fully clear. Using a murine model of dermal infection with Leishmania parasites, we observe a transient neutrophil influx in draining lymph nodes despite sustained recruitment to the infection site. Cell-tracking experiments, together with intravital two-photon microscopy, indicate that neutrophil recruitment to draining lymph nodes occurs minimally through lymphatics from the infected dermis, but mostly through blood vessels via high endothelial venules. Mechanistically, neutrophils do not respond to IL-1ß or macrophage-derived molecules. Instead, they are guided by the C5a-C5aR1 axis, using L-selectin and integrins, to extravasate into the draining lymph node parenchyma. We also report that C5, the C5a precursor, is locally produced in the draining lymph node by lymphatic endothelial cells. Our data establish and detail organ-specific mechanisms of neutrophil trafficking.

Novel insights into negative pressure wound healing from an in situ porcine perspective.

Negative pressure wound therapy (NPWT) is used clinically to promote tissue formation and wound closure. In this study, a porcine wound model was used to further investigate the mechanisms as to how NPWT modulates wound healing via utilization of a form of NPWT called the vacuum-assisted closure. To observe the effect of NPWT more accurately, non-NPWT control wounds containing GranuFoam™ dressings, without vacuum exposure, were utilized. In situ histological analysis revealed that NPWT enhanced plasma protein adsorption throughout the GranuFoam™, resulting in increased cellular colonization and tissue ingrowth. Gram staining revealed that NPWT decreased bacterial dissemination to adjacent tissue with greater bacterial localization within the GranuFoam™. Genomic analysis demonstrated the significant changes in gene expression across a number of genes between wounds treated with non-NPWT and NPWT when compared against baseline tissue. However, minimal differences were noted between non-NPWT and NPWT wounds, including no significant differences in expression of collagen, angiogenic, or key inflammatory genes. Similarly, significant increases in immune cell populations were observed from day 0 to day 9 for both non-NPWT and NPWT wounds, though no differences were noted between non-NPWT and NPWT wounds. Furthermore, histological analysis demonstrated the presence of a foreign body response (FBR), with giant cell formation and encapsulation of GranuFoam™ particles. The unique in situ histological evaluation and genomic comparison of non-NPWT and NPWT wounds in this pilot study provided a never-before-shown perspective, offering novel insights into the physiological processes of NPWT and the potential role of a FBR in NPWT clinical outcomes.

Role of chain architecture in the solution phase assembly and thermoreversibility of aqueous PNIPAM/silyl methacrylate copolymers.

Stimuli-responsive polymers functionalized with reactive inorganic groups enable creation of macromolecular structures such as hydrogels, micelles, and coatings that demonstrate smart behavior. Prior studies using poly(N-isopropyl acrylamide-co-3-(trimethoxysilyl)propyl methacrylate) (P(NIPAM-co-TMA)) have stabilized micelles and produced functional nanoscale coatings; however, such systems show limited responsiveness over multiple thermal cycles. Here, polymer architecture and TMA content are connected to the aqueous self-assembly, optical response, and thermo-reversibility of two distinct types of PNIPAM/TMA copolymers: random P(NIPAM-co-TMA), and a 'blocky-functionalized' copolymer where TMA is localized to one portion of the chain, P(NIPAM-b-NIPAM-co-TMA). Aqueous solution behavior characterized via cloud point testing (CPT), dynamic light scattering (DLS), and variable-temperature nuclear magnetic resonance spectroscopy (NMR) demonstrates that thermoresponsiveness and thermoreversibility over multiple cycles is a strong function of polymer configuration and TMA content. Despite low TMA content (≤2% mol), blocky-functionalized copolymers assemble into small, well-ordered structures above the cloud point that lead to distinct transmittance behaviors and stimuli-responsiveness over multiple cycles. Conversely, random copolymers form disordered aggregates at elevated temperatures, and only exhibit thermoreversibility at negligible TMA fractions (0.5% mol); higher TMA content leads to irreversible structure formation. This understanding of the architectural and assembly effects on the thermal cyclability of aqueous PNIPAM-co-TMA can be used to improve the scalability of responsive polymer applications requiring thermoreversible behavior, including sensing, separations, and functional coatings.

Myeloid antigen-presenting cell niches sustain antitumor T cells and license PD-1 blockade via CD28 costimulation.

The mechanisms regulating exhaustion of tumor-infiltrating lymphocytes (TIL) and responsiveness to PD-1 blockade remain partly unknown. In human ovarian cancer, we show that tumor-specific CD8+ TIL accumulate in tumor islets, where they engage antigen and upregulate PD-1, which restrains their functions. Intraepithelial PD-1+CD8+ TIL can be, however, polyfunctional. PD-1+ TIL indeed exhibit a continuum of exhaustion states, with variable levels of CD28 costimulation, which is provided by antigen-presenting cells (APC) in intraepithelial tumor myeloid niches. CD28 costimulation is associated with improved effector fitness of exhausted CD8+ TIL and is required for their activation upon PD-1 blockade, which also requires tumor myeloid APC. Exhausted TIL lacking proper CD28 costimulation in situ fail to respond to PD-1 blockade, and their response may be rescued by local CTLA-4 blockade and tumor APC stimulation via CD40L.

Investigation of human adipose-derived stem-cell behavior using a cell-instructive polydopamine-coated gelatin-alginate hydrogel.

Hydrogels can be fabricated and designed to exert direct control over stem cells' adhesion and differentiation. In this study, we have investigated the use of polydopamine (pDA)-treatment as a binding platform for bioactive compounds to create a versatile gelatin-alginate (Gel-Alg) hydrogel for tissue engineering applications. Precisely, pDA was used to modify the surface properties of the hydrogel and better control the adhesion and osteogenic differentiation of human adipose-derived stem cells (hASCs). pDA enabled the adsorption of different types of bioactive molecules, including a model osteoinductive drug (dexamethasone) as well as a model pro-angiogenic peptide (QK). The pDA treatment efficiently retained the drug and the peptide compared to the untreated hydrogel and proved to be effective in controlling the morphology, cell area, and osteogenic differentiation of hASCs. Overall, the findings of this study confirm the efficacy of pDA treatment as a valuable strategy to modulate the biological properties of biocompatible Gel-Alg hydrogels and further extend their value in regenerative medicine.

A New Micromonospora Strain with Antibiotic Activity Isolated from the Microbiome of a Mid-Atlantic Deep-Sea Sponge.

To tackle the growing problem of antibiotic resistance, it is essential to identify new bioactive compounds that are effective against resistant microbes and safe to use. Natural products and their derivatives are, and will continue to be, an important source of these molecules. Sea sponges harbour a diverse microbiome that co-exists with the sponge, and these bacterial communities produce a rich array of bioactive metabolites for protection and resource competition. For these reasons, the sponge microbiota constitutes a potential source of clinically relevant natural products. To date, efforts in bioprospecting for these compounds have focused predominantly on sponge specimens isolated from shallow water, with much still to be learned about samples from the deep sea. Here we report the isolation of a new Micromonospora strain, designated 28ISP2-46T, recovered from the microbiome of a mid-Atlantic deep-sea sponge. Whole-genome sequencing reveals the capacity of this bacterium to produce a diverse array of natural products, including kosinostatin and isoquinocycline B, which exhibit both antibiotic and antitumour properties. Both compounds were isolated from 28ISP2-46T fermentation broths and were found to be effective against a plethora of multidrug-resistant clinical isolates. This study suggests that the marine production of isoquinocyclines may be more widespread than previously supposed and demonstrates the value of targeting the deep-sea sponge microbiome as a source of novel microbial life with exploitable biosynthetic potential.

A new view of macula densa cell microanatomy.

Although macula densa (MD) cells are chief regulatory cells in the nephron with unique microanatomical features, they have been difficult to study in full detail due to their inaccessibility and limitations in earlier microscopy techniques. The present study used a new mouse model with a comprehensive imaging approach to visualize so far unexplored microanatomical features of MD cells, their regulation, and functional relevance. MD-GFP mice with conditional and partial induction of green fluorescent protein (GFP) expression, which specifically and intensely illuminated only single MD cells, were used with fluorescence microscopy of fixed tissue and live MD cells in vitro and in vivo with complementary electron microscopy of the rat, rabbit, and human kidney. An elaborate network of major and minor cell processes, here named maculapodia, were found at the cell base, projecting toward other MD cells and the glomerular vascular pole. The extent of maculapodia showed upregulation by low dietary salt intake and the female sex. Time-lapse imaging of maculapodia revealed highly dynamic features including rapid outgrowth and an extensive vesicular transport system. Electron microscopy of rat, rabbit, and human kidneys and three-dimensional volume reconstruction in optically cleared whole-mount MD-GFP mouse kidneys further confirmed the presence and projections of maculapodia into the extraglomerular mesangium and afferent and efferent arterioles. The newly identified dynamic and secretory features of MD cells suggest the presence of novel functional and molecular pathways of cell-to-cell communication in the juxtaglomerular apparatus between MD cells and between MD and other target cells.NEW & NOTEWORTHY This study illuminated a physiologically regulated dense network of basal cell major and minor processes (maculapodia) in macula densa (MD) cells. The newly identified dynamic and secretory features of these microanatomical structures suggest the presence of novel functional and molecular pathways of cell-to-cell communication in the juxtaglomerular apparatus between MD and other target cells. Detailed characterization of the function and molecular details of MD cell intercellular communications and their role in physiology and disease warrant further studies.

Rapid tumor vaccine using Toll-like receptor-activated ovarian cancer ascites monocytes.

BACKGROUND: Novel therapeutic strategies in ovarian cancer (OC) are needed as the survival rate remains dismally low. Although dendritic cell-based cancer vaccines are effective in eliciting therapeutic responses, their complex and costly manufacturing process hampers their full clinical utility outside specialized clinics. Here, we describe a novel approach of generating a rapid and effective cancer vaccine using ascites-derived monocytes for treating OC. METHODS: Using the ID8 mouse ovarian tumor model and OC patient samples, we isolated ascites monocytes and evaluated them with flow cytometry, Luminex cytokine and chemokine array analysis, ex vivo cocultures with T cells, in vivo tumor challenge and T cell transfer experiments, RNA-sequencing and mass spectrometry. RESULTS: We demonstrated the feasibility of isolating ascites monocytes and restoring their ability to function as bona fide antigen-presenting cells (APCs) with Toll-like receptor (TLR) 4 lipopolysaccharide and TLR9 CpG-oligonucleotides, and a blocking antibody to interleukin-10 receptor (IL-10R Ab) in the ID8 model. The ascites monocytes were laden with tumor antigens at a steady state in vivo. After a short 48 hours activation, they upregulated maturation markers (CD80, CD86 and MHC class I) and demonstrated strong ex vivo T cell stimulatory potential and effectively suppressed tumor and malignant ascites in vivo. They also induced protective long-term T cell memory responses. To evaluate the translational potential of this approach, we isolated ascites monocytes from stage III/IV chemotherapy-naïve OC patients. Similarly, the human ascites monocytes presented tumor-associated antigens (TAAs), including MUC1, ERBB2, mesothelin, MAGE, PRAME, GPC3, PMEL and TP53 at a steady state. After a 48-hour treatment with TLR4 and IL-10R Ab, they efficiently stimulated oligoclonal tumor-associated lymphocytes (TALs) with strong reactivity against TAAs. Importantly, the activated ascites monocytes retained their ability to activate TALs in the presence of ascitic fluid. CONCLUSIONS: Ascites monocytes are naturally loaded with tumor antigen and can perform as potent APCs following short ex vivo activation. This novel ascites APC vaccine can be rapidly prepared in 48 hours with a straightforward and affordable manufacturing process, and would be an attractive therapeutic vaccine for OC.